The traditional Chinese medicine Bupleuri radix (chaihu) is the dried roots of Bupleurum chinense and Bupleurum scorzonerifolium and many adulterants exist because of the differences in traditional understanding, medication habits and seed resources. Therefore, rapid and accurate identification methods for Bupleurum (Apiaceae) seeds are required. We analyzed the internal transcribed spacer (ITS) sequences of five common Bupleurum species to detect variations in them, including B. chinense, B. scorzonerifolium, B. marginatum var. stenophyllum, B. falcatum and B. smithii var.parvifolium. Based on single nucleotide polymorphisms (SNPs) in the ITS region, we designed five specific primer pairs and used these primers in an allele-specific PCR technique to establish a robust molecular identification method. The neighbor-joining (NJ) tree of ITS sequences showed that five Bupleurum species formed their own monophyly. Five specific primer pairs were designed and integrated into a specific PCR master mix and cycling conditions. The primer pair of BCF/R8 for B. chinense could amplify a specific identification band of 429 bp and the minimum detection limit of the 5 g mixture was about 5%; BSF/R11 for B. scorzonerifolium could amplify a specific 464 bp band and the minimum detection limit was about 5%; BMSF/R1 for B. marginatum var. stenophyllum could amplify a specific 344 bp band and the minimum detection limit was about 1%; BFF/R7 for B. falcatum could amplify a specific 137 bp band and the minimum detection limit was about 1%; BSPF/R1 for B. smithii var. parvifolium could amplify a specific 390 bp band. Subsequent analysis proved the reliable accuracy and good practicability of the five specific identification primers, indicating that the allele-specific primer PCR identification method can quickly identify Bupleurum seeds. The method elaborated in this study has the advantages of simple operation, good accuracy and high efficiency.

中文翻译：

---

通过基于ITS序列的等位基因特异性PCR鉴定柴胡种子。

柴胡（柴胡）是柴胡和柴胡的干燥根，由于传统认识，用药习惯和种子资源的差异，存在许多掺假品。因此，需要一种快速，准确的鉴别柴胡种子的方法。我们分析了五个常见柴胡种类的内部转录间隔区（ITS）序列，以检测其中的变异，包括chinensis，scorzonerifolium，marginatum var。甜叶菊，B。falcatum和B. smithii var.parvifolium。基于ITS区中的单核苷酸多态性（SNP），我们设计了五对特异性引物，并在等位基因特异性PCR技术中使用了这些引物，以建立可靠的分子鉴定方法。ITS序列的相邻结（NJ）树表明，柴胡5个物种形成了自己的一字形。设计了五个特定的引物对，并整合到特定的PCR预混液和循环条件中。B. chinense的BCF / R8引物对可扩增429 bp的特异性鉴定条带，并且5 g混合物的最低检测限为5％。BSF / R11可用于蝎形芽孢杆菌的扩增，扩增一条464 bp的特异性条带，最低检出限为5％。B.marginatum变种的BMSF / R1。甜叶菊可扩增一个344 bp的特异性条带，最低检出限约为1％。B. falcatum的BFF / R7可以扩增137 bp的特异性条带，最小检出限约为1％。B.smithii变种的BSPF / R1。细小叶可以扩增特定的390 bp条带。随后的分析证明了五种特异性鉴定引物的准确性和实用性，表明等位基因特异性引物PCR鉴定方法可以快速鉴定柴胡种子。本研究阐述的方法具有操作简单，准确度高，效率高的优点。